The present invention relates to methods for preparing cell lines that contain artificial chromosomes, to methods for isolation of the artificial chromosomes, targeted insertion of heterologous DNA into the chromosomes, isolation of the chromosomes, and delivery of the chromosomes to selected cells and tissues. Also provided are cell lines for use in the methods, and cell lines and chromosomes produced by the methods.
Several viral vectors, non-viral, and physical delivery systems for gene therapy have been developed [see, e.g., Mitani et al. (1993) Trends Biotech. 11:162-166]. The presently available systems, however, have numerous limitations, particularly where persistent, stable, or controlled gene expression is required. These limitations include: (1) size limitations because there is a limit, generally on order of about ten kilobases [kB], at most, to the size of the DNA insert [gene] that can be accepted by viral vectors, whereas a number of mammalian genes of possible therapeutic importance are well above this limit, especially if all control elements are included; (2) the inability to specifically target integration so that random integration is required which carries a risk of disrupting vital genes or cancer suppressor genes; (3) the expression of randomly integrated therapeutic genes may be affected by the functional compartmentalization in the nucleus and are affected by chromatin-based position effects; (4) the copy number and consequently the expression of a given gene to be integrated into the genome cannot be controlled. Thus, improvements in gene delivery and stable expression systems are needed [see, e.g., Mulligan (1993) Science 260:926-932].
In addition, safe and effective gene therapy methods and vectors should have numerous features that are not assured by the presently available systems. For example, a safe vector should not contain DNA elements that can promote unwanted changes by recombination or mutation in the host genetic material, should not have the potential to initiate deleterious effects in cells, tissues, or organisms carrying the vector, and should not interfere with genomic functions. In addition, it would be advantageous for the vector to be non-integrative, or designed for site-specific integration. Also, the copy number of therapeutic gene(s) carried by the vector should be controlled and stable, the vector should secure the independent and controlled function of the introduced gene(s); and the vector should accept large (up to Mb size) inserts and ensure the functional stability of the insert.
The limitations of existing gene delivery technologies, however, argue for the development of alternative vector systems suitable for transferring large [up to Mb size or larger] genes and gene complexes together with regulatory elements that will provide a safe, controlled, and persistent expression of the therapeutic genetic material.
At the present time, none of the available vectors fulfill these requirements. Some of these characteristics, however, are possessed by chromosomes. Thus, an artificial chromosome would be an ideal vector for gene therapy, as well as for production of gene products that require coordination of expression of numerous genes or that are encoded by large genes, and other uses. Artificial chromosomes for expression of heterologous genes in yeast are available, but construction of a mammalian artificial chromosome has not been achieved. Such construction has been hindered by the lack of an isolated, functional, mammalian centromere and uncertainty regarding the requisites for its production and stable replication. Unlike in yeast, there are no selectable genes in close proximity to a mammalian centromere, and the presence of long runs of highly repetitive pericentric heterochromatic DNA makes the isolation of a mammalian centromere using presently available methods, such as chromosome walking, virtually impossible. Other strategies are required for production of mammalian artificial chromosomes, and some have been developed. For example, U.S. Pat. No. 5,288,625 provides a cell line that contains an artificial chromosome, a minichromosome, that is about 20 to 30 megabases. Methods provided for isolation of these chromosomes, however, provide preparations of only about 10-20% purity. Thus, development of alternative artificial chromosomes and perfection of isolation methods as well as development of more versatile chromosomes and further characterization of the minichromosomes is required to realize the potential of this technology.
Therefore, it is an object herein to provide mammalian artificial chromosomes and methods for introduction of foreign DNA into such chromosomes. It is also an object herein to provide methods for introduction of the artificial mammalian chromosome into selected cells, and to provide the resulting cells, as well as transgenic animals and plants that contain the artificial chromosomes. It is also an object herein to provide methods for gene therapy and expression of gene products using artificial chromosomes. It is a further object herein to provide methods for constructing species-specific artificial chromosomes.
Mammalian artificial chromosomes [MACs] are provided. Also provided are artificial chromosomes for other higher eukaryotic species, such as insects and fish, produced using the MACS are provided herein. Methods for generating and isolating such chromosomes. Methods using the MACs to construct artificial chromosomes from other species, such as insect and fish species are also provided. The artificial chromosomes are fully functional stable chromosomes. Two types of artificial chromosomes are provided. One type, herein referred to as SATACs [satellite artificial chromosomes] are stable heterochromatic chromosomes, and the another type are minichromosomes based on amplification of euchromatin.
Artificial chromosomes permit targeted integration of megabase pair size DNA fragments that contain single or multiple genes. Thus methods using the MACs to introduce the genes into cells, animals and tissues are also provided. The artificial chromosomes with integrated heterologous DNA are to be used in methods of gene therapy, in methods of production of gene products, particularly products that require expression of multigene biosynthetic pathways, and also are intended for delivery into the nuclei of germane cells, such as embryo-derived stem cells [ES cells] for production of transgenic animals.
Mammalian artificial chromosomes provide extra-genomic specific integration sites for introduction of genes encoding proteins of interest and permit megabase size DNA integration so that, for example, genes encoding an entire metabolic pathway or a very large gene, such as the cystic fibrosis [CF; xcx9c600 kb] gene, several genes, such as a series of antigens for preparation of a multivalent vaccine, can be stably introduced into a cell. Vectors for targeted introduction of such genes, including the tumor suppressor genes, such as p53, the cystic fibrosis transmembrane regulator gene [CFTR], anti-HIV ribozymes, such as an anti-HIV gag ribozyme, into the artificial chromosomes also provided.
The chromosomes provided herein are generated by introducing heterologous DNA that includes DNA encoding a selectable marker into cells, preferably a stable cell line, growing the cells under selective conditions, and identifying from among the resulting clones those that include chromosomes with more than one centromere or that have chromosomes that are fragments of chromosomes that had more than one centromere. The amplification that produces the additional centromere occurs in cells that contain chromosomes in which the heterologous DNA has integrated near the centromere in the pericentric region of the chromosome. The selected clonal cells are then used to generate artificial chromosomes.
In preferred embodiments, the DNA with the selectable marker that is introduced includes sequences that target it to the pericentric region of the chromosome. For example, vectors, such as pTEMPUD, which includes such DNA specific for mouse satellite DNA, are provided. Also provided are derivatives of pTEMPUD that specifically target human satellite sequences. Upon integration, these vectors can induce the amplification.
Artificial chromosomes are generated by culturing the cells with the dicentric chromosomes under conditions whereby the chromosome breaks to form a minichromosome and formerly dicentric chromosome. The artificial chromosomes [the SATACs] are generated, not from the minichromosome fragment as, for example, in U.S. Pat. No. 5,288,625, but from the fragment of the formerly dicentric chromosome.
Among the MACs provided herein are the SATACs, which are primarily made up of repeating units of short satellite DNA and are fully heterochromatic, so that absent insertion of heterologous or foreign DNA, the chromosomes do not contain genetic information. They can thus be used as xe2x80x9csafexe2x80x9d vectors for delivery of DNA to mammalian hosts because they do not contain any potentially harmful genes.
In addition to MACs methods for generating euchromatic minichromsomes and the use thereof are also provided herein. Methods for generating one type of MAC the minichromosome, previously described in U.S. Pat. No. 5,288,625, and the use thereof for expression of heterologous DNA are provided. Cell lines containing the minichromosome and the use thereof for cell fusion are also provided.
In one embodiment, a cell line containing the mammalian minichromosome is used as recipient cells for donor DNA encoding a selected gene or multiple genes. The donor DNA is linked to a second selectable marker and is targeted to and integrated into the minichromosome. The resulting chromosome is transferred by cell fusion into an appropriate recipient cell line, such as a Chinese hamster cell line [CHO]. After large scale production of the cells carrying the engineered chromosome, the chromosome is isolated. In particular, metaphase chromosomes are obtained, such as by addition of colchicine, and they are purified from the cell lysate. These chromosomes are used for cloning, sequencing and for delivery of heterologous DNA into cells.
Also provided are SATACs of various sizes that are formed by repeated culturing under selective conditions and subcloning of cells that contain chromosomes produced from the formerly dicentric chromosomes. These chromosomes are based on repeating units 7.5 to 10 Mb referred to herein as megareplicons, that are tandem blocks of satellite DNA flanked by heterologous non-satellite DNA. Amplification produces a tandem array of identical chromosome segments [each called an amplicon] that contain two inverted megareplicons bordered by heterologous [xe2x80x9cforeignxe2x80x9d] DNA. Repeated cell fusion, growth on selective medium and/or BrdU [5-bromodeoxyuridine] treatment or other genome destabilizing reagent or agent, such as ionizing radiation, including X-rays, and subcloning results in cell lines that carry stable heterochromatic or partially heterochromatic chromosomes, including a 150-200 Mb xe2x80x9csausagexe2x80x9d chromosome, a 500-1000 Mb gigachromosome, a stable 250-400 Mb megachromosome and various smaller stable chromosomes derived therefrom. These chromosomes are based on these repeating units and can include heterologous DNA that is expressed.
Thus methods for producing MACs of both types are provided. These methods are applicable to any higher eukaryotic cell, including mammals, insects and plants.
The resulting chromosomes can be purified by methods provided herein to provide vectors for introduction of the heterologous DNA into selected cells for production of the gene product encoded by the heterologous DNA, for production of transgenic animals and plants or for gene therapy. Vectors for chromosome fragmentation are provided. These vectors will be used to produce an artificial chromosome that contains a megareplicon, a centromere and two telomeres and will be between about 10 Mb and about 60 Mb, preferably between about 10 Mb-15 Mb and 30 Mb. Such artificial chromosomes may be produced by other methods. Isolation of the 7.5 Mb [or 15 Mb amplicon containing two 7.5 Mb inverted repeats] or a 30 Mb multimer thereof should provide a stable chromosomal vector that can be manipulated in vitro.
In addition, methods and vectors for fragmenting the minichromosomes and SATACs are provided. Such methods and vectors can be used for in vivo generation of smaller stable artificial chromosomes. Methods for reducing the size of the MACs to generate smaller stable self-replicating artificial chromosomes are also provided.
Methods and vectors for targeting heterologous DNA into the artificial chromosomes are also provided as are methods and vectors for fragmenting the chromosomes to produce smaller but stable and self-replicating artificial chromosomes. Vectors for targeted introduction of heterologous DNA into artificial chromosomes are provided.
The chromosomes are introduced into cells to produce stable transformed cell lines or cells, depending upon the source of the cells. Introduction is effected by any suitable method including, but not limited to electroporation, direct uptake, such as by calcium phosphate [see, e.g., Wigler et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76:1373-1376; and Current Protocols in Molecular Biolog, Vol. 1, Wiley Inter-Science, Supplement 14, Unit 9.1.1-9.1.9 (1990)]. precipitation, uptake of isolated chromosomes by lipofection, by microcell fusion [see, EXAMPLES, see, also Lambert (1991) Proc. Natl. Acad. Sci. U.S.A. 88:5907-5911, U.S. Pat. No. 5,396,767] or other suitable method. The resulting cells can be used for production of proteins in the cells. The chromosomes can be isolated and used for gene delivery.
Methods for isolation of the chromosomes based on the DNA content of the chromosomes, which differs from the authentic chromsomes are provided.
These artificial chromosomes can be used in gene therapy, gene product production systems, production of humanized organs, production of transgenic plants and animals, including invertebrates, vertebrate, reptiles and insects, any organism or device that would employ chromosomal elements as information storage vehicles, and also for analysis and study of centromere function, for the production of artificial chromosome vectors that can be constructed in vitro, and for the preparation of species- specific artificial chromosomes. The artificial chromosomes can be introduced into cells using microinjection, cell fusion, microcell fusion, electroporation, electrofusion, projectile bombardment, calcium phosphate precipitation, site-specific targeting and other such methods. Cells particularly suited for use with the artificial chromosomes include, but are not limited to plant cells, particularly tomato, arabidopsis, and others, insect cells, including silk worm cells, insect larvae, fish, reptiles, amphibians, arachnids, mammalian cells, embryonic stem cells, embryos and cells for use in methods of genetic therapy, such as lymphocytes that are used in methods of adoptive immunotherapy and nerve or neural cells. Thus methods of producing gene products and transgenic animals and plants are provided. Also provided are the resulting transgenic animals and plants.
Exemplary cell lines that contain these chromosomes are also provided.
Methods for preparing artificial chromosomes for particular species and for cloning centromeres are also provided. In particular, a method for cloning a centromere from an animal or plant by preparing a library of DNA fragments that contain the genome of the plant or animal, introducing the each of the fragments into a mammalian satellite artificial chromosome [SATAC] that contains a centromere from a different species, generally a mammal, from the selected plant or animal, generally a non-mammal, and a selectable marker. The selected plant or animal is one in which the mammalian species centromere does not function. Each of the SATACs is introduced into the cells, which are grown under selective conditions, and cells with SATACs are identified. Such SATACS should contain a centromere encoded by the DNA from the library.
Also provided are libraries in which the relatively large fragments of DNA are contained on artificial chromosomes.
Transgenic animals, invertebrates and vertebrates, plants and insects, fish, reptiles, amphibians, arachnids and mammals are also provided. Of particular interest are transgenic animals that express genes that confer resistance or reduce susceptibility to disease. Since multiple genes can be introduced on a MAC, a series of genes encoding an antigen can be introduced, which up expression will serve to immunize [in a manner similar to a multivalent vaccine] the host animal against the diseases for which exposure to the antigens provide immunity or some protection.
Methods for cloning centromeres, such as mammalian centromeres, are also provided. In particular, in one embodiment, a library composed of fragments of the SATACs are cloned into YACs [yeast artificial chromosomes] that include a detectable marker, such as DNA encoding tyrosinase and then introduced into mammalian cells, such as albino mouse embryos. Mice produced from YACs that include a centromere that functions in mammals will express the detectable marker. Thus, if albino mice will be pigmented or have regions of pigmentation.